Plasma preparation

Mouse blood was collected from orbital venous plexus or inferior vena cava, with 3.8% sodium citrate (1:9) added. The samples were then centrifuged at 5000g for 10 min to obtain platelet-poor plasma (PPP) which was stored at – 70 °C. Assay results with the combined plasma of WT mice was taken as standard for FVIII activity.

Synthesis of siRNAs

The siRNA sequences were designed based on transcripts in NCBI (LMAN1-Homo NM_005570, Lman1-Mouse NM_027400.3, MCFD2-Homo sapiens NM_139279.6, MCFD2-Mouse NM_176808). Then they were screened with the Dual-Luciferase reporter assay. The sense and antisense single strands of the optimal ones were chemically modified at selected sites with either 2′-O-methyl or 2′-F nucleosides, and sense strand was conjugated to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc), promoting targeted delivery to the liver. The GalNAc-siRNAs were synthesized with the help of the GenePharma Company (Suzhou, China).

Dual-Luciferase reporter assay and cell immunofluorescence assay

293T cells were purchased from cell bank of Chinese Academy of Sciences (Shanghai, China). Fragments containing the binding sites of the siRNAs were synthesized and sub-cloned into the psiCHECK-2 plasmid (Promega, USA; C8021). Cells were seeded in 96-well plate at a density of 1 × 105 cells/well. After 24 h, cells were transfected with the siRNAs using lipofectamine 2000 (Invitrogen, USA; 53,566) according to the manufacturer’s protocol. Reporter plasmids were transfected into the cells simultaneously. Luciferase activity was determined at 24 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega, USA; E2940). For tracking the GalNAc-siRNAs, HepaG2 cells (Procell, China, CL-0103) were transfected with FAM-labeled GalNAc-siRNAs and examined 24 h later using a confocal fluorescence microscope at a wavelength of 488nm.

Quantitative reverse transcription PCR (RT-qPCR)

To detect the transcripts of LMAN1 or MCFD2, total RNA was extracted from mice liver or transfected HEPAG2 cells using TRIzol (BBI, China; B511311). The RNA was converted to cDNA using PrimeScript RT Master Mix (Takara, Japan; RR036A) to amplify various partial fragment of LMAN1 or MCFD2 mRNA. Primer sequences are shown in Supplementary Table S1. Quantitative real-time PCR was performed using AceQ Universal SYBR qPCR Master Mix (Vazyme, China; Q511-02) with the Applied Biosystems 7500 Real-Time PCR System. Results were normalized with GAPDH RNA, and the fold change was determined using the 2-ΔΔCT method.

Western blotting

To assess the levels of LMAN1 and MCFD2, proteins were isolated from mouse livers using Protein Extraction kit (BBI, China; B510005). Total cellular lysates (30 mg/lane) were separated on 10% or 15% SDS-PAGE gels. The membranes were blocked and incubated overnight with primary antibodies, followed by incubation with secondary antibodies for 2 h. The target bands were visualized in an imaging analyzer, and the gray value of each band was quantified by an ImageJ software. The expression level of GAPDH was used as reference. Primary antibodies are as follows: anti-LMAN1 rabbit monoclonal (Cell Signaling Technology, USA; clone E2B6H), Anti-MCFD2 rabbit polyclonal (Absin, China, abs151162), Anti-FVIII mouse polyclonal (Abcam, USA, ab171825), anti-GAPDH antibody (Cell Signaling Technology, USA, clone 14C10). Secondary antibodies are as follows: rabbit anti-mouse IgG (BBI, China; D110098) and goat anti-rabbit IgG (BBI, China; D110058).

Coagulation assays

Collected mouse plasma was tested for prothrombin time (PT) and activated partial thromboplastin time (APTT) using assay kits (Sysmex, China; R9055 and R9051, respectively) and four-channel semi-automated coagulation analyzer STart (Diagnostica Stago, France). The activity of FVIII in mouse plasma was detected using Coamatic Factor VIII kit (K822585, Chromogenix, Italy) following the protocol provided with the kit.

Pharmacokinetics of siRNAs in mice

Female mice (C57BL/6, 6–8 weeks old, n = 5/group) supplied by Vital River Laboratory Animal Technology Co., Ltd (Zhejiang, China) were subcutaneously administered with GalNAc-LMAN1 or GalNAc-MCFD2 at 10 mg/kg. At 12 h and on days 7 and 14 post administration, the mice were sacrificed and tissue samples, including liver, heart, spleen, lung, muscle, kidney, and serum, were collected. The drug levels in these organs were accessed using Stem Loop-qPCR assays [14,15,16] (GenePharma, Suzhou, China) according to the provided instruction. The mice were perfused with saline before tissue collection. The sequences of the primers used for the Stem-Loop qPCR assays are shown in Fig. 3B. Because the GalNAc of each siRNA acts similarly, LMAN1-181 and MCFD2-36 were tested as examples.

Pharmacodynamics of siRNAs in mice

Female mice (C57BL/6, 6–8 weeks old, n = 6/group) were subcutaneously administered with either 0.9% saline or GalNAc-siRNAs at 3mg/kg or 10 mg/kg. 7 days after the 3mg/kg single dose injection, plasma samples from inferior vena cava were collected to measure FVIII activity. Livers were collected, and the expression of LMAN1 and MCFD2 mRNA was examined by RT-qPCR, and protein levels were examined by western blotting. In a separate set of experiment, mice received three doses of siRNA at 10mg/kg on days 1, 4 and 7. Plasma samples from retro-orbital venous plexus were collected at various times, and the FVIII and were measured with APTT assay (see Fig. 5A for the schedule of injection and blood collection).

Tail bleeding test

The mice in the drug administration group and the normal control group were subjected to a mouse tail bleeding test 10 days after the first administration. Mice (male, 8–10 weeks) were anesthetized with 1% sodium pentobarbital solution and then placed in the prone position. The 2-mm distal portion of the tail was removed with a scalpel and the rest of the tail was immediately immersed in a 15 ml centrifuge tube containing 0.9% saline that was preheated to 37 °C in a water bath. The tail was kept vertical and below the body level. Each animal was monitored for 20 min. Blood loss was determined by hemoglobin content after red cell lysis with 2% acetic acid and was expressed in microliters of blood per gram (g) mouse body weight assuming a hematocrit of 46%. If bleeding did not stop within 20 min, the experiment was terminated within 20 min according to the approved animal protocol to avoid death during the procedure.

Statistical analysis

All values were expressed as means plus or minus standard deviation (SD). Comparisons between the groups were performed using independent-samples t-test and two-way ANOVA test with GraphPad Prism 9.0 software. P values of less than 0.05 were considered statistically significant.

Patients

All F5F8D patients reported in this study were from hematology clinic of the First Affiliated Hospital of Soochow University. The recently identified proband and her family members were sampled for genetic analysis. The DNAs were extracted using a whole blood DNA reagent kit (QIAGEN, Hilden, Germany). Genomic DNA analysis using a gene sequencing platform for hemostasis and thrombosis (HAT platform) that included 199 genes related to hemorrhage, thrombi, and platelets [17]. This study was approved by the Medical Ethical Committee of Soochow University. Informed consent was obtained from all patients and healthy controls in accordance with the Declaration of Helsinki.